We have previously shown that inhibition of the actin activation of vertebrate skeletal myosin subfragment-1 (S-1) ATP hydrolysis by troponin-tropomyosin in the absence of Ca-2+ is due to the reduction of the rate of a step in the ATP hydrolysis cycle which occurs subsequent to the binding of HMM to actin. Others have reported that the two-headed fragment of myosin (HMM) shows, in the presence of ATP and in the absence of Ca-2+ a large reduction in affinity to actin (in addition to the kinetic type of inhibition that is observed for S-1) provided that the regulatory light chains of HMM are intact. In contrast, we have failed to detect any large difference in the binding constant to actin (plus or minus) Ca-2+ using preparations of HMM with 90% intact light chains. Double binding experiments showed that in the absence of Ca-2+ more than 75% of the total HMM population binds to actin-troponin-tropomyosin with about half the affinity as in the presence of Ca-2+. Thus we must conclude that the regulatory system in skeletal muscle does not operate by weakening binding of either S-1 or HMM to actin in the absence of Ca-2+. We have also studied the regulatory system of mollusan muscle which does not require troponin-tropomyosin. Actin activation of the HMM ATPase of this system occurs when Ca-2+ binds directly to the HMM. In this system the binding constant of HMM-ATP to actin in the absence of Ca-2+ is 1/5 as strng as the binding constant in the presence of Ca-2+. However, like the vertebrate regulatory system the major effect of removing Ca-2+ isto reduce the rate of a process which occurs subsequent to the binding of HMM to actin.